GABAB Receptor 1
GABA (γ-aminobutyric acid) is an endogenous neurotransmitter in the central and peripheral nervous systems. Receptors for GABA have traditionally been divided into GABAA and GABAB receptor subtypes. GABAB receptors (for a review see Kerr, D. I. B. and Ong, J. (1995) Pharmac. Ther. vol. 67, pp. 187-246) belong to the superfamily of G-protein coupled receptors. GABAB receptor agonists are described as being of use in the treatment of CNS disorders, such as muscle relaxation in spinal spasticity, cardiovascular disorders, asthma, gut motility disorders such as irritable bowel syndrome and as prokinetic and anti-tussive agents. GABAB receptor agonists have also been disclosed as useful in the treatment of emesis (WO 96/11680) and reflux disease (WO 98/11885).
The cloning of the cDNA encoding the rat GABAB receptors splice isoforms GABABR1a and GABABR1b is disclosed by Kaupmann et al. (1997) Nature, vol. 386, 239-246. The mature rat GABABR1b differed from GABABR1a in that the N-terminal 147 residues were replaced by 18 different residues. It was presumed that the rat GABABR1a and -b receptor variants are derived from the same gene by alternative splicing.
The cloning of the cDNA encoding the human GABAB receptor GABABR1b is disclosed in WO 97/76675 .
The cloning of the human GABAB receptor 1 gene and the elucidation of the exon-intron organization is in part or fully disclosed in PCT/SE98/01947, in EMBL HS271M21 (GenBank AL031983), EMBL AJ010170 to AJ010191, in Peters, H C et al., Neurogenetics 2; 47-54 (1998) and in Goei, V L et al. Biological Psychiatry. 44; 659-66 (1998). The human GABAB receptor 1 gene consists of 23 exons, spanning over a distance of 30 kb (FIG. 1). The elucidation of the gene organization revealed that the human GABABR1a and GABABR1b are splice variants encoded by a single gene. The GABABR1a and GABABR1b isoforms are differentially expressed, at least in the rat (Kaupmann et al. (1997) Nature, vol. 386, 239-246). The physiological consequences of multiple GABAB receptor 1 splice isoforms has not yet been determined, but their existence constitute an opportunity for the development of specific pharmaceutical agents.
GABAB Receptor 2
Based on its homology with the mammalian GABABR1 cDNA, a second member of the GABAB receptor family was identified (Jones, K A et al., Nature 396; 674-679 (1998), White, J A et al., Nature 396; 679-682 (1998), Kaupmann, K et al., Nature 396; 683-687 (1998), WO 99/20751). The corresponding protein, GABABR2, forms heteromers with GABABR1a and R1b, resulting in cell surface expression of a functional GABA B receptor (Kuner, R et al. Science 283, 74-77 (1999)). At least in recombinant expression systems, GABABR1 and R2 coexpression is necessary for the formation of a functional GABAB receptor. Jones et al. (Nature 396; 674-679 (1998)) disclosed that a GABABR1: GABABR2 stochiometry of 1:1 is an optimal ratio for successful cell surface expression of a ligand binding and functional GABAB receptor. Thus, modulating GABABR1 expression could alter the stochiometry between GABABR1 and other interacting proteins and be a means to regulate signaling through GABAB receptors and thereby interfere with various physiological processes.
Transcriptional Regulation
Gene regulation is mediated by specific DNA elements in the promoter that directs binding of transcription factors, which thereby mediate transcription of the gene. Eukaryotic transcription factors can be divided in two main groups i) basal transcription factors that interact with promoter sequences proximal to the start of transcription, thereby initiating transcription upon recruitment of RNA polymerase II and ii) transcription factors that bind to specific distal promoter elements, thereby modulating the transcription upon contact with the basal transcription machinery. The DNA sequence that directs the start of transcription in most eukaryotic genes is the TATA-box, which is often located approximately 30 base pairs upstream from the start of transcription. However, the TATA-box is not a prerequisite for initiation of transcription as there are many promoters, including the GABAB R1 promoters described in this study, that lack a TATA-box. A fundamental physiological process in the eukaryotic organism is that cells can communicate with their environment and respond to extracellular stimuli through signaling molecules, such as hormones and growth factors. The final event for such signaling is the binding of transcription factors to specific distal promoter elements leading to for example up-regulated or tissue specific gene expression. Because of their regulatory role, signaling molecules are putative targets for screening of therapeutic agents. The presence of two distinct and differentially regulated promoters within the human GABAB receptor 1 gene, disclosed in this patent application, makes it possible to screen for therapeutic agents selectively regulating expression of GABAB receptor 1a and 1b-type splice isoforms.
Indications
Compounds which are modulators of GABAB receptor 1 transcription are potentially useful in the treatment of disorders which are related to neurally-controlled physiological responses regulated by GABAB receptors, e.g. CNS disorders such as muscle relaxation in spinal spasticity, Alzheimer's disease and other dementias, psychiatric and neurological disorders such as depression, anxiety and epilepsy, cardiovascular disorders, asthma, gut motility disorders such as irritable bowel syndrome, emesis and reflux disease. In some humans, the lower esophageal sphincter (LES) is prone to relaxing more frequently than in other humans. As a consequence, fluid from the stomach can pass into the esophagus since the mechanical barrier is temporarily lost at such times, an event hereinafter referred to as “reflux”.
Gastro-esophageal reflux disease (GERD) is the most prevalent upper gastrointestinal tract disease. Current therapy has aimed at reducing gastric acid secretion, or by reducing esophageal acid exposure by enhancing esophageal clearance, lower esophageal sphincter tone and gastric emptying. The major mechanism behind reflux has been considered to depend on a hypotonic lower esophageal sphincter. However, recent research (e.g. Holloway & Dent (1990) Gastroenterol. Clin. N. Amer. 19, 517-535) has shown that most reflux episodes occur during transient lower esophageal sphincter relaxations (TLESR), i.e. relaxations not triggered by swallows. It has also been shown that gastric acid secretion usually is normal in patients with GERD. Consequently, there is a need for compounds which reduce the incidence of TLESR and thereby prevent reflux.